Southern Blot
Southern blotting is a mainstay laboratory technique for detecting specific DNA sequences among DNA fragments. Although used less frequently in the modern laboratory, similar techniques inspired by the Southern blot, like the Western blot and Northern blot, which identify proteins and RNA molecules respectively, have become indispensable. Southern blotting is used to identify target sequences within a DNA sample. This is a powerful tool with many applications. Southern blotting can be used to identify DNA sequences within a restriction digest (for forensic, environmental, etc. purposes), to locate homologous sequences within a sample, and to locate genes within a restriction digest, which could lead to mapping the structure of a genome. Southern blotting has been an important technique in clinical labs as well. For example, Coros et al used Southern blotting to identify the IS6110 sequence, once thought to exist exclusively in Mycobacterium tuberculosis, within Mycobacterium smegmatis, ''indicating that horizontal gene transfer may have occurred between these two species and the IS6110 sequence could no longer be used as a diagnostic indicator for ''M. tuberculosis ''(Coros 2008). Method The DNA fragments are separated by gel electrophoresis and visualized for later comparison. The DNA is then denatured and transferred from the gel to a nitrocellulose membrane by capillary action. Some modern methods call for a nylon membrane instead of nitrocellulose. The gel rests on top of a sponge soaked in neutral saline solution (saline sodium citrate) and beneath the nitrocellulose membrane and a stack of dry filter paper or paper towels. As the saline solution is driven upwards towards the paper towels, it carries the DNA with it. The nitrocellulose membrane is impermeable to nucleic acids and "catches" the DNA as the solution passes through. DNA is then fixed to the membrane by baking or UV radiation before being immersed in a solution containing radioactive RNA probes that are complimentary to the target sequence. The membrane is then washed in saline solution and visualized, most commonly by X-ray. The entire process from gel electrophoresis to visualization of the hybridized fragments can be done in as little as five days (Southern 2006). Once visualized, the hybridized membrane is compared to the initial visualization of the gel electrophoresis to identify which fragment of DNA contains the target sequence. Development Southern blotting was developed by Edwin Southern at the University of Edinburgh. Southern had developed the technique as early as 1973 but did not get the method published until 1975. At the time, Southern was working in collaboration with Peter Ford, who was attempting to isolate and sequence the genes for 5S ribosomal RNA in two types of frog cells, without the benefit of modern sequencing technology. Southern came up with the idea of hybridizing the DNA on the gel with radioactive RNA as a way to identify the 5S RNA within the frog cell samples (Gitschier 2013). Southern's originally illustrated his technique using ''E. coli ''and ''Xenopus laevis ''ribosomal DNA (a known sequence) cut from a larger DNA sample with restriction enzymes. Southern ran the DNA on an agarose gel then used the capillary action technique to transfer the DNA from the gel to a nitrocellulose membrane. He then exposed the membrane to 32P-tagged RNA probes complimentary to the ribosomal DNA sequences and visualized them with ultraviolet radiation. The accuracy of Southern's technique was confirmed by the appearance of radioactivity within the fragments known to contain the complimentary ribosomal DNA sequences, indicating the 32P-tagged probes had hybridized with their complimentary sequences (Southern 1975). References Coros A, DeConno E, Derbyshire K. 2008. [http://jb.asm.org/content/190/9/3408.full.pdf IS6110, a ''Mycobacterium tuberculosis complex-specific insertion sequence, is also present in the genome of Mycobacterium smegmatis, ''suggestive of gene transfer between Mycobacterial species.] ''Journal of Bacteriology. 190(9): 3408. Gitscher J. 2014. A problem solved: An interview with Sir Edward Southern. PLoS Genetics. PMCID: 3597496. Southern EM. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. ''Journal of Molecular Biology. ''98, 503-517. Southern EM. 2006. Southern blotting. ''Nature Protocols. ''1(2), 518-524.